Currently, blood transfusion is mainly supported by blood donation in the medical field, but numerous efforts and costs are required for its collection, examination of infections and the like, and storage in order to assure its supply. In order to supply sufficient amounts of blood in the medical and research fields, a method of inducing a mature erythrocyte from a cultured hematopoietic stem cell within a short time and efficiently is required. If a technology for culturing a hematopoietic stem cell from a totipotent or pluripotent stem cell including an iPS cell, an ES cell, and the like is developed and practically applied in future, the cell can be artificially cultured in all of the steps and stable supply of blood with a low risk of infection becomes possible, which highly contributes to medical procedures. Further, industrial culture of blood in a large amount becomes possible, and thus blood production with a low cost and its development as a medical industry can be expected.
An erythrocyte is an important blood component which carries oxygen, but the erythrocyte itself cannot proliferate because it has no nucleus. Thus, in vivo, an erythroid precursor cell, which is a cell at a stage prior to the erythrocyte, proliferates and matures through “enucleation” which is deprived of the nucleus.
Therefore, in order to produce the erythrocyte in a large amount, mass culture of the erythroid precursor cell is needed, and thus, establishment of a “cell line (erythroid precursor cell line)” is needed.
Further, in the research for hematopoiesis, the group of Ihle et al. reported that “SOCS3 was essential for hematopoiesis in the fetal liver” (see Non Patent Literature 1).
In addition, the group of Nagata et al. reported that “fetal DNase II knockout mice were not able to produce a mature erythrocyte and experienced severe anemia, and macrophage DNase II in the fetal liver was important as a support for the erythrocyte differentiation” (see Non Patent Literature 2).
Meanwhile, the group of RIKEN established an erythroid precursor cell line from a murine ES cell and enabled the culture of an erythroid precursor cell in a large amount in vitro (see Non Patent Literature 3).
As the mass culture is available, a method of directly transfusing an erythroid precursor cell into a body is also possible. However, safety aspects such as canceration are largely concerned because the precursor cell is nucleated. In actuality, it is necessary to develop an induction method including further differentiating the precursor cell for enucleation to obtain a mature erythrocyte with no nucleus.
Further, the method of producing a mature erythrocyte utilizing cord blood reported by Miharada et al. (Non Patent Literature 4) discloses that “the generation of a mature enucleated erythrocyte from an erythroid precursor cell required culture for 20 days and the ratio of a final enucleated cell was 77%.”
In the method of producing a mature erythrocyte, no factor for inducing enucleation is added to a medium, the erythroid precursor cell is differentiated by a differentiation-inducing factor added in an early phase of the culture, and the enucleation is induced as natural maturation associated therewith.
As described above, several methods of producing an erythroid precursor cell have already been known, but a technology for enucleating a nucleated erythrocyte within a short time and with high efficiency during the process has been little known. However, there is the following report on a method of adding no factor for inducing enucleation to a medium (see Patent Literature 1).
Patent Literature 1 discloses a “method of inducing enucleation by culturing a hematopoietic stem cell in the absence of erythropoietin.” However, an enucleation rate is 1 to 5% on Day 11 and 65 to 80% on Day 15. That is, the induction of enucleation takes a long time and the enucleation rate is also insufficient.
From the above-mentioned background, a nucleated erythrocyte remains at a ratio of about 20 to 30% in an erythrocyte preparation obtained by a currently available production method, and further the administration of such erythrocyte in vivo has a risk of canceration, which is problematic.
In addition, in future, the induction of a nucleated erythrocyte in a large amount from a human iPS or ES cell becomes possible, and as a result, it is expected that a method of inducing enucleation of an erythrocyte within a short time is demanded.